Introduction: Cholestasis is a pathophysiological condition caused by different etiologies such as infection, autoimmunity, metabolic disorders, and genetic predisposition. Studies have shown that cholestasis reduces the proliferation of spermatogenic cells and induces apoptosis, leading to the contraction and deformation of seminal tubes, as well as preventing the maturation of germ cells. Spermatogonial stem cells (SSCs) are essential for self-renewal and differentiation during spermatogenesis. Murine undifferentiated spermatogonial cells and SSCs express high levels of Thy-1 and all SSCs in rat testicles are Thy-1 positive cells. Kubo and colleagues demonstrated that the expression of CD90 or Thy-1 in rodents implies the presence of SSCs in testes. In current study we conducted a study to investigate the expression of Thy-1 as a reliable marker for SSCs.
Methods: Eight adult male Wistar rats were divided into two groups named as control and cholestatic (n=4 each). To induce obstructive cholestasis their common bile duct was closed by surgery and the expression of Thy-1 in testis tissue of cholestatic and control rats, was assessed by quantitative real time PCR (qRT-PCR) technique. First, the primer sequence for Thy-1 and GAPDH was designed and then, the total RNA was extracted from the tissues and converted to cDNA. The synthesized cDNA was used to carry out the reverse transcription reaction and the relative expression of Thy-1 was determined using R=2^(- (ΔΔCT)) formula. To carry out immunofluorescent staining, heat antigen retrieval was first performed using hydrochloric acid (HCl) and then tissue sections were treated with 0.3% Triton X-100 in phosphate-buffered saline (PBS) for 30 minutes to permeabilize the membrane of cells to allow the passage of the antibody to the cell interior. Next, tissue sections were washed with PBS to remove PBS-T and specimens were then treated with 10% goat serum for 30 minutes to block non-specific bindings. Afterward, goat serum was washed once with PBS. The slides were treated with antibodies against Thy-1 for 12 hours. Nuclear labeling was done with DAPI and the slides were observed using fluorescent microscope Olympus model. The ImageJ software was used to quantify the fluorescent intensity. The total cellular fluorescence (TCF) was calculated by the following formula: TCF = integrated density − (area of selected cells × mean fluorescence of background readings).
Results: The results of qRT-PCR showed that the expression of Thy-1 was markedly (p-value=0.05) reduced in the cholestasis group compared with the control group. The results of expression of Thy-1 protein in testicular tissues indicated that the expression of Thy-1 was substantially declined in the cholestasis group as compared with the control group. So, the total cellular florescence of Thy-1 in the cholestasis group was 35.47 ± 1.590 while it was 52.03 ± 1.686 in the control group, showing a significant difference (p-value=0.002) between the two groups.
Conclusion: The expression of gene and protein of Thy-1 was significantly lower in the cholestasis group than that of the control group. The reduction in the expression of this specific SSC marker verified the decreased number of SSCs. This may be one of the ways that cholestasis can damage testicular tissue and impair spermatogenesis. The current study shows one of the important side effects of cholestasis on testicular tissue and its consequent infertility-related problems in men.